Chromatography Forum

Hello all,

I'm analyzing a 20 KDa protein by Reverse phase using a Jupiter C4 column. The problem is that the main peak has a persistent tail and contaminants actually co-elute during this tailing (confirmed by SDS-PAGE of fractionated run).



Current conditions:
- Jupiter C4 column 250mm*4.6mm 5µm particle (new column)
- Buffer A: 5% ACN in water 0.1%TFA
- Buffer B: 95% ACN in water 0.1%TFA
- Gradient for 0% to 100% in 30 mns
- Temperature: 40ºC
-100µl injection
-Flow rate: 1ml/min
- Sample buffer: Buffer A

Already tried without success:
- Using propanol instead of ACN
- Higher column temperature
- C8 column
- slower gradient (more tailing)
- Different buffer sample
- Injecting 1/10 of the amount of protein.

All this attempts produced a peak with a similar shape.

I was inclined to try using TEA or acetic acid or formic acid instead of TFA.

Does anyone have any suggestions as what I could try to improve the peak shape and separation?

Many thanks for your attention,

Joana

Hi Joana,

Prepare the following eluents:
A – 0.1 M sodium phosphate pH 2.5 (adjusted before addition of ACN) add 5 % ACN.
B – 85 % ACN in water.
And try again.
Important! Remember to wash the column thoroughly (with 50 % ACN/water for 1 hour and then with the new eluents, running a couple of gradients) before the new conditions.

Best Regards

Hi Joana -

I recommend using a TFA gradient, but doing a 10-75%B.
If you're lucky - you'll notice a trend (which will help you for
further optimization).

If you look at Imtakt's RP protein data (using Intrada WP-RP) - it
usually starts with 10-20% organic:

Interferon: http://www.silvertonesciences.com/files/TI263E.pdf
Hemoglobin: http://www.silvertonesciences.com/files/TI272E.pdf
Bovine Serum (injected directly into the column):
http://www.silvertonesciences.com/files/TI274E.pdf

WP-RP is a novel design but it's reversed phase mode - so optimization principles should be
similar as for other reversed phase columns (though the results may be different).

Thanks Bryan and Danko for your quick replies.

I have already tried starting with a higher organic percent but the peak profile was still the same.

I will now try using the phosphate buffer at pH 2.5. I will report the results back later.

Thanks